Cosolutes and the Stability of a Domain-Swapped Dimer

Undergraduates: Gerardo Perez Goncalves, Alex J. Guseman Gary J. Pielak

Faculty Advisor: Gary Pielak
Department: Chemistry

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The cellular interior is a complex environment where the concentration of macromolecules exceeds 300 g/L. Under these crowded conditions, proteins experience hard-core repulsions and chemical interactions with cytoplasmic components that are not present in buffered alone. Hard core repulsions arise from steric repulsions and stabilize to proteins. Chemical interactions can be stabilizing if repulsive and destabilizing if attractive. Studies of these interactions have broadened our understanding of protein chemistry in cells, but most use simple monomeric proteins. However, proteins rarely work alone, and protein-protein interactions give rise to complex protein architectures. A variant (T2Q/L5V/F30V/Y33F/A34F) of the B1 domain of protein G forms a domain swapped homodimer. I labeled the sole tryptophan of the domain with fluorine labeling and used 19F nuclear magnetic resonance (NMR) to quantify the effect of cosolutes on the dimer dissociation constant. Macromolecular cosolutes stabilize the dimer, suggesting the importance of hard-core repulsions. Experiments with some of the monomers indicate that chemical interactions are destabilizing. The effects of biologically relevant protein crowders show that they work through an intricate mix of hard-core repulsions and chemical interactions.